Reverse Transcription

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Reverse Transcription
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Central Dogma
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• Normal transcription involves synthesis of RNA
  from DNA.
• Reverse transcription is the transcription of
  single stranded RNA into double stranded DNA
• With the help of the enzyme Reverse
  Transcriptase.

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• Reverse Transcriptase also known as RNA directed
  DNA Polymerase
• - DNA Nucleotidyl transferase (RNA directed)
• - Revertase
• Reverse Transcriptase was discovered by Howard
  Temin and Baltimore in 1970 independently
• - shared Nobel Prize in Physiology or Medicine in
  1975 for their discovery.

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• Reverse transcriptase common in Retrovirus.
• - HIV
• M-MLV (Moloney Murine Leukemia Virus)
• AMV (Avian Myeloblastosis Virus)
• Reverse Transcriptase enzyme includes two
  activity DNA polymerase and RNase H

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Retrovirus Replication Cycle
ds DNA
host DNA
Provirus
Packaging into virus budding
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Antiviral drug

• Zidovudine
• Lamivudine
• Tenfovir

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• Typical retrovirus has three or four genes.
• Gag-pol- env
• Retrovirus are called strand because viral RNA
  itself code for protein products.
• Reverse transcriptase enzyme code for proteins
  are called strand
• R Redundant or repeating seq 10-80
• U5 80 100
• U3 170-1350
• Like DNA polymerase, Reverse transcriptase
  requires primers.
• tRNA of the host is Primer

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MECHANSIM OF RETROVIRUS REPLICATION
1. A Retrovirus specific cellular tRNA hybridizes
with a complementary region called PBS (Primer
Binding Sites)
2. Reverse Transcriptase (RT) starts at this
binding site and copies RNA into a single strand
of complementary DNA. A DNA segment is extended
from tRNA based on the sequence of the retroviral
genomic RNA
3. The viral R and U5 sequences are removed by
RNase H.
LTR Long Terminal Repeat Left LTR - Redundant
sequence R 5 untranslated region (U5) Right
LTR - Redundant sequence R 3 untranslated
region (U3)
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4. First jump DNA hybridizes with the remaining
R sequence at the 3' end.
5. A DNA strand is extended from the 3' end.
6. Most viral RNA is removed by RNase H
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7. A second DNA strand is extended from the viral
RNA.
8. Both tRNA and the remaining viral RNA are
removed by RNase H.
9. Second jump The PBS region of the second
strand hybridizes with the PBS region of the
first strand.
10. Extension on both DNA strands. 
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• Reverse transcriptase has a high error rate when
  transcribing RNA into DNA as unlike DNA
  Polymerase, since it has no proof reading ability
• This high error rate allows mutations to
  accumulate
• The commercially available RT produced by Promega
  are quoted high error rates in range of one in
  17,000 bp for AMV and one in 30,000 bases for
  M-MLV

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cDNA

• Reverse Transcriptase can use single stranded RNA
  into DS DNA cDNA.
• cDNA library A set of clones representing as
  many as possible of the mRNAs in a given cell
  type at given time.
• In the preparation cDNA library, mRNA is
  extracted, purified, and treated with the enzyme
  reverse transcriptase.
• Complementary DNA (cDNA) analogs of the isolated
  mRNA are thereby obtained.
• Since mature mRNA contains no introns or
  regulatory regions, a cDNA library composed of
  coding regions.

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